In this report period, we carried on our long-standing research line, development of anti human immunodeficiency virus (HIV) drugs. We pursued three major lines of research: Project 1 - Identification and development of new antiviral agents active against HIV-1; Project 2 - Study of CC-chemokine/CCR5 interactions and development of CCR5 inhibitors as potential therapeutics for therapy of AIDS; and Project 3 - Study of the mechanisms of emergence of drug-resistant HIV-1 variants. Project 1: We have identified novel protease inhibitors containing 3(R),3a(S),6a(R)-bis tetrahydrofuranyl urethane (bis-THF) in collaboration with Professor Arun Ghosh of University of Illinois at Chicago. It is of note that UIC-00317/TMC114, one of the bis-THF congeners, has proceeded to Phase IIb Clinical trials in european countries. Continuing further collaboration with Dr. Ghosh and his group, we are developing more effective and resistant HIV-"repellant" protease inhibitors containing unique active components.Project 2: Considering that certain CXCR4/CCR5 inhibitors, if successfully developed, would "resist" to the emergence of resistant virus because the target for CXCR4/CCR5 inhibitors is indeed a cellular apparatus, therefore, HIV-1 cannot use the same strategy to acquire resistance to RTIs and PIs: "to change itself". We thus redirected our attentions from the NRTI- and PI related research lines and started our efforts to identify such entry inhibitors. In collaboration with scientists of Ono Pharmaceutical Co., Ltd., which is based in Osaka, Japan, we identified a new class of CCR5 inhibitors, spirodiketopiperazine (SDP) derivatives. SDP derivatives bind to CCR5, block CC-chemokine(CC)/CCR5 interactions, and inhibit HIV-1 infection of CCR5+ cells (Maeda & Mitsuya, J. Biol. Chem. 276:35194, 2001). One such CCR5 inhibitor aplaviroc (AVC: AK602/ONO4128/GW873140), exerts potent activity against a wide spectrum of laboratory and primary R5-HIV-1 isolates including multi-drug resistant HIVMDR (IC50 values of 0.2-0.6 nM), which is associated with its high CCR5-binding affinity (KD values of 2 nM) and potent inhibition of CCR5-gp120 binding. AVC is now under multiple phase III clinical trials in the United States. Previously reported CCR5 inhibitors tested (e.g., Sch-C, TAK779) fully blocked HIV infection as well as CC/CCR5 binding, while AVC, despite its much greater anti-HIV activity, partially suppressed the interactions at high concentrations (Maeda & Mitsuya, J. Virol. 78:8654, 2004). We also characterized the binding profile of AVC by using CHO cells expressing various mutant CCR5 species whose amino acid(s) was substituted in the extracellular loops (ECL) and transmembrane (TM) domain of CCR5. The data suggest that AVC's binding site is located in a hydrophobic pocket in the proximity of the interface between the second ECL (ECL-2B) and the upper TM domain. Several mutations in the pocket turned out to decrease the binding of HIV gp120 and all CC chemokines examined to CCR5, suggesting that the hydrophobic pocket is critical for CCR5's ligand-specific conformational changes induced by CC-chemokines and gp120. AVC, once bound to CCR5+ human cells, remained on the cell surface for >9 hr after thorough washing, and blocked R5-HIV infection upon delayed HIV-1 exposure. When intraperitoneally administered twice daily to R5-HIV-1JRFL-infected hu-PBM-NOD-SCID mice, AK602 successfully suppressed HIV-1 replication and blocked CD4+ T-cell decrease (Nakata & Mitsuya, J. Virol. 79: 2087, 2005). Phase I clinical trial in healthy individuals showed that AK602 was safe and well tolerated with no QTc prolongation. Receptor occupancy studies demonstrate prolonged receptor binding to CCR5 and suggest QD or BID dosing. In an early phase II-10 day monotherapy study enrolling HIV-1 viremia in patients with AIDS, AVC demonstrated potent antiviral activity (mean 1.66 log decrease in viral load at nadir, Lalezari et al., ICAAC 2004) .